PROJECT SUMMARY The discovery of new and effective treatments for coronary heart disease (CHD), the leading cause of death in the world, requires the identification of novel disease mechanisms. Using genome-wide association studies (GWASs) in large population cohorts and exome sequencing in individuals with an unusual lipid pattern that we have termed ?familial combined hypolipidemia??extremely low blood lipids across the board but otherwise healthy?we identified the ANGPTL3 (angiopoietin-like 3) gene as being linked to both blood triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels. More recently, we have demonstrated that naturally occurring loss-of-function mutations in ANGPTL3 are not only linked to lower TG and LDL-C levels but also to substantial protection against CHD. Our work therefore recommends ANGPTL3 as a compelling therapeutic target, perhaps rivalling or even surpassing the PCSK9 gene. ANGPTL3, a protein exclusively synthesized in hepatocytes and secreted into the bloodstream, is well established to inhibit lipoprotein lipase, increasing blood TG levels. The mechanism by which ANGPTL3 increases blood LDL-C levels remains to be determined. In preliminary studies, we have begun to ascertain the effects of naturally occurring ANGPTL3 missense variants using an Angptl3 knockout mouse model complemented with a physiological level of the human ANGPTL3 gene. We have found that specific missense variants have differing effects on TG and cholesterol levels in mice, providing us with a means to distinguish between the physiological consequences of TG modulation and LDL-C modulation in humans. We have also found that some missense variants inhibit ANGPTL3?s effects on both TG and cholesterol levels, providing us with variants that we can attempt to individually introduce into human hepatocytes in vivo with genome editing for therapeutic purposes. Our specific aims are: (1) to assess the physiological effects of natural ANGPTL3 missense variants in mice; (2) to assess the physiological effects of natural ANGPTL3 missense variants in humans; and (3) to use base editing, a new type of genome editing, to introduce ANGPTL3 missense variants into human hepatocytes in vivo. We will achieve these aims by employing a novel mouse model that can interrogate ANGPTL3 variants? effects on TG and LDL-C levels; by taking advantage of the Penn Medicine BioBank cohort, in which ~12,000 individuals have been exome-sequenced?more than 500 of whom have ANGPTL3 missense variants?and have given consent to be called back for potential participation in human physiological studies; and by building on our expertise in base editing, paired with a chimeric liver-humanized mouse model, that will allow us to interrogate the efficacy and safety of introducing ANGPTL3 variants into human hepatocytes in vivo?a novel therapeutic approach that has the potential to yield a ?vaccine? against CHD.